DESCRIPTION: This grant application is aimed at establishing a link between the fine structure of the gap junction channel wall and channel permselectivity. The investigators propose to study the properties of rat Cx26, Cx32, Cx37, Cx40, Cx43 and Cx46 when expressed in transfected N2A cells. They will analyze the spread of anionic fluorescent probes via estimations of three basic parameters: cytoplasmic diffusion coefficient, junctional membrane permeability and leak permeability. The second specific aim is to transfect mouse N2A cells with mutant versions of the connexins studied under Specific aim 1, and to evaluate the same permeability parameters. Through these studies, the investigators propose to locate the site(s) that constitute the anionic filter. The mutagenesis analysis will focus on the four transmembrane domains, and the two extracellular loops. Under specific aim three, the applicants will use the dual whole cell patch clamp technique to verify the existence of gap junction channels and, if channels are found, to determine the unitary conductance and the cation/anion permeability properties of the mutant under study. Finally, under specific aim four, the investigators propose to characterize a relation between channel properties and dye transfer. The latter may allow for determination of permeability of large anions at the channel level.